2 7 dichlorofluorescein dcf

Manufactured by Merck Group

Sourced in United States

2',7'-dichlorofluorescein (DCF) is a fluorescent dye commonly used as a laboratory reagent. It is a highly sensitive indicator for the detection of reactive oxygen species (ROS) in various biological systems. DCF exhibits increased fluorescence upon oxidation, making it a useful tool for monitoring oxidative stress and cellular redox status.

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Lab products found in correlation

9 protocols using 2 7 dichlorofluorescein dcf

Dispersing ZnO Nanoparticles for Cell Assays

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Zinc oxide nanoparticles (ZnO NPs) with a diameter less than 100 nm were dispersed in phosphate-buffered saline (PBS) and subjected to ultrasonication for 5 min to prevent aggregation prior to cell treatment. The ZnO NP was then prepared in the culture medium at various concentrations. ZnO NP and 2,7-Dichlorofluorescein (DCF) were purchased from Sigma-Aldrich (St. Louis, MO, USA). FluoZin™-3, AM was purchased from Thermo Fisher Scientific (Carlsbad, CA, USA).

Kim B., Kim G., Jeon H.P, & Jung J. (2024). Lipidomics Analysis Unravels Aberrant Lipid Species and Pathways Induced by Zinc Oxide Nanoparticles in Kidney Cells. International Journal of Molecular Sciences, 25(8), 4285.

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Antioxidant Evaluation of Valtrate Compounds

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HPLC-grade methanol and acetonitrile were purchased from Fisher Scientific (Fair Lawn, NJ, USA). A Milli-Q water purification system (Millipore, San Diego, CA, USA) was used to further purify distilled water. Other materials included cisplatin (CP), N, N- Dimethylformamide (DMF), 2,2’-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt (ABTS), 1,1-diphenyl-2-picrylhydrazyl radical 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH), 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethylsulfoxide (DMSO), butylated hydroxytoluene (BHT), 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) and 2,7-dichlorofluorescein (DCF), all of which were purchased from Sigma Aldrich Co. (St. Louis, MO, USA). Annexin V-fluorescein isothiocyanate (Annexin V-FITC) and a propidium iodide (PI) kit were obtained from BD (San Jose, CA, USA). Valtrate (lot: 111840–201402), acevaltrate (lot: 111841–201401) and 1-β acevaltrate were obtained from the National Institutes for Food and Drug Control of China. The analytical reagent-grade sodium hydroxide, hydrochloric acid and hydrogen peroxide (H₂O₂, 30%) used in this study were purchased from Beijing Chemical Works (Beijing, China).

Wang F., Zhang Y., Wu S., He Y., Dai Z., Ma S, & Liu B. (2017). Studies of the structure-antioxidant activity relationships and antioxidant activity mechanism of iridoid valepotriates and their degradation products. PLoS ONE, 12(12), e0189198.

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Investigating Cellular Pathways Modulation

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Rotenone, sulforaphane, dimethyl sulfoxide (DMSO), 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) and 2′,7′-dichlorofluorescein (DCF) were purchased from Sigma (St. Louis, MO, USA); CMC: (Nacalai Tesque, Kyoto, Japan); Protease and phosphatase inhibitor mini tables (Thermo Scientific, USA); RIPA lysis buffer was purchased from Beyotime, China. The following antibodies were used: phospho-S6K (Thr389), 4E-BP1, Cleaved-caspase-3, LC3A/B (Cell Signalling Technology, Beverly, MA, USA); phospho-mTOR (Ser2448), mTOR (all from sigma); p70S6K; Nrf2, heme oxygenase-1 (HO-1); NAD(P)H dehydrogenase, quinone 1/(NQO1) and monoclonal mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Tyrosine hydroxylase (TH) (Millipore, Billerica, MA). Dulbecco’s Modified Eagle’s Medium (GIBCO, Gaithersburg, MD, USA). Other chemicals were provided by local commercial sources and were of analytical grade quality.

Zhou Q., Chen B., Wang X., Wu L., Yang Y., Cheng X., Hu Z., Cai X., Yang J., Sun X., Lu W., Yan H., Chen J., Ye J., Shen J, & Cao P. (2016). Sulforaphane protects against rotenone-induced neurotoxicity in vivo: Involvement of the mTOR, Nrf2, and autophagy pathways. Scientific Reports, 6, 32206.

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Mitochondrial ROS and Mass Analysis

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Intracellular and mitochondrial ROS, as well as mitochondrial mass, were measured using flow cytometric analysis. 2 × 10⁵ cells per condition were incubated for 15 min at 37 °C with 2 μM of 2′,7′-dichlorofluorescein (DCF) (Sigma Aldrich, St. Louis, MO) to detect intracellular ROS, 5 μM MitoSOX™ (Molecular Probes, Eugene, OR, USA) to label superoxide, specifically produced by mitochondria, and 100 nM MitoTracker Green (Molecular Probes, Invitrogen Corp., Carlsbad, CA, USA), a labeling mitochondria probe. After washing in PBS, 10⁴ events for each sample were acquired using a Navios flow cytometer and analysed with Kaluza Analysis 1.3 software.

Tataranni T., Mazzoccoli C., Agriesti F., De Luca L., Laurenzana I., Simeon V., Ruggieri V., Pacelli C., Della Sala G., Musto P., Capitanio N, & Piccoli C. (2019). Deferasirox drives ROS-mediated differentiation and induces interferon-stimulated gene expression in human healthy haematopoietic stem/progenitor cells and in leukemia cells. Stem Cell Research & Therapy, 10, 171.

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Oxidative stress assay protocol

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Purpurin (purity ≥ 85.0%), Curcumin (purity ≥ 98.0%), L-glutathione reduced (GSH, purity ≥ 98.0%), L-glutathione oxidized (GSSG, purity ≥ 98.0%), L-buthionine-sulfoximine (BSO, purity ≥ 97.0%), N-acetyl-L-cysteine (NAC, purity ≥ 99.0%), tert-butyl hydroperoxide (TBHP, 70% in water) 2′,7′-dichlorofluorescein (DCF, purity ~90%) and 2′,7′-dichlorofluorescein diacetate (H2DCF-DA, purity ≥ 97.0%) were obtained from Sigma Aldrich (Zwijndrecht, The Netherlands). All buffer salts, including citric acid, tri-sodium citrate dihydrate, di-sodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate monohydrate and formic acid, and acetonitrile for Liquid Chromatography/Mass Spectrometry assay, were also obtained from Sigma Aldrich (Zwijndrecht, The Netherlands). Methanol of analytical grade was ordered from Fisher Scientific (Loughborough, United Kingdom). Dimethyl sulfoxide (DMSO) was purchased from Acros Organics (Geel, Belgium). Dulbecco’s Modified Eagle Medium with Ham’s Nutrient Mixture F-12 (1:1) (DMEM/F12), DMEM/F12 without phenol red, trypsin 0.05% EDTA, nonessential amino acids (NEAA) and phosphate-buffered saline pH 7.4 (PBS), geneticin (G418), penicillin/streptomycin, were purchased from Gibco (Carlsbad, CA, USA). Fetal bovine serum (FBS) was purchased from Invitrogen (Breda, The Netherlands).

Ren Q., Bakker W., Wesseling S., Bouwmeester H, & Rietjens I.M. (2023). On the Role of ROS and Glutathione in the Mode of Action Underlying Nrf2 Activation by the Hydroxyanthraquinone Purpurin. Antioxidants, 12(8), 1544.

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ROS production in Aci44 and Aci46 under colistin, panduratin A, and combination treatments

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The productions of reactive oxygen species (ROS) by Aci44 and Aci46 after treatment with colistin, panduratin A, or panduratin-colistin combination were measured using 2′-7′-Dichlorofluorescein (DCF) (Sigma-Aldrich, USA)⁴⁹. Aci44 and Aci46 at 10⁶ CFU/mL in PBS were pre-incubated for 30 min with 5 µM DCF at 37 °C, then treated with colistin, panduratin A, or panduratin-colistin combination. Aci44 and Aci46 were treated with four conditions: (1) 0.05 × MIC (0.1 mg/L colistin for Aci44 and 1.6 mg/L colistin for Aci46) colistin combined with 5 µM panduratin A, (2) colistin alone, (3) 5 µM panduratin A alone, and (4) untreated (or CAMHB). The treated strains were incubated at 37 °C for 1 h in 96-black well plate. Twenty µM H₂O₂ treatment was used as a positive control. Fluorescence intensity was measured using Clariostar plus microplate reader (BMG Labtech, Germany) at excitation wavelength of 480 nm and emission wavelength of 530 nm.

Thadtapong N., Chaturongakul S., Napaswad C., Dubbs P, & Soodvilai S. (2024). Enhancing effect of natural adjuvant, panduratin A, on antibacterial activity of colistin against multidrug-resistant Acinetobacter baumannii. Scientific Reports, 14, 9863.

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Quantifying Cellular ROS Production

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To assess ROS production, cells were incubated with 2′7′-dichlorofluorescein (DCF; 10 μM; Sigma) for 30 min prior to analysis by flow cytometry using the LSR Fortessa instrument (BD, San Jose, CA, USA). Cells without added DCF probes were used as a control of non-specific signal. Hoechst 33258 (5 μg/ml; Invitrogen, Carlsbad, CA, USA) was added to cells prior to the measurements to exclude dead cells from the analysis.

Hubackova S., Davidova E., Boukalova S., Kovarova J., Bajzikova M., Coelho A., Terp M.G., Ditzel H.J., Rohlena J, & Neuzil J. (2020). Replication and ribosomal stress induced by targeting pyrimidine synthesis and cellular checkpoints suppress p53-deficient tumors. Cell Death & Disease, 11(2), 110.

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Graphene Oxide Nanomaterial Characterization

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Graphene oxide water dispersion (0.4 wt% concentration) was purchased from Graphenea Inc., (Gipuzkoa, Spain). TETRA-HPRG (Ac-(GHHPH)₄G-NH₂ with the addition of 6-Fam (6-carboxyfluorescein) were purchased from Caslo (Kongens Lyngby, Denmark). Phosphate buffered saline purchased by Sigma-Aldrich (St. Louis, MO, USA). Ultrapure Milli-Q water was used (18.2 mΩ∙cm at 25 °C, Millipore, Burlington, MA, USA). RPMI 1640, Dulbecco’s modified eagle medium (DMEM)-F12, fetal bovine serum (FBS), penicillin streptomycin solution and amphotericin solution for cell cultures. 2′,7′-dichlorofluorescein (DCF) were purchased from Sigma-Aldrich (St. Louis, MO, USA). MitoSOX™ red mitochondrial superoxide indicator and 2′-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5′-bi-1H-benzimidazole trihydrochloride trihydrate (Hoechst33342) were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Verde V., Longo A., Cucci L.M., Sanfilippo V., Magrì A., Satriano C., Anfuso C.D., Lupo G, & La Mendola D. (2020). Anti-Angiogenic and Anti-Proliferative Graphene Oxide Nanosheets for Tumor Cell Therapy. International Journal of Molecular Sciences, 21(15), 5571.

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Evaluating Porphyrin Nanoparticles for ROS

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Human bladder cancer 5637 cells (ATCC®, Manassas, VA) were seeded into 96-well plates overnight. After treatment with various concentrations of PNPs and NPs for 4 hours, free drugs were washed and cells were lysed with 100 μl of lysis buffer for 30 minutes with shake. Fluorescence was measured by ELISA reader (Molecular devices, Sunnyvale, CA).
For intracellular reactive oxygen species (ROS) productions, 5637 cells were treated with 10 μg/ml PNPs (Pyropheophorbide a : 2μg/ml) for 2 hours and washed by PBS for 3 times in suspension. Cells were then loaded with 10μM 2′,7′-dichlorofluorescein (DCF) (Sigma) for 30 minutes followed by 4.2 J/cm² light treatment (Omnilux New-U LED panel with 635 nm light, Clifton Park, NY). ROS production was then analyzed by flow cytometry. Methods for assessment of the tissue level of ROS production were described previously[19 ].
To specifically confirm the signet oxygen generation in vitro, we incubated singlet oxygen sensor green (SOSG, Sigma) with different concentrations of PNPs with or without SDS. SOSG and porphyrin fluorescence were measured by ELISA reader.

Lin T.Y., Li Y., Liu Q., Chen J.L., Zhang H., Lac D., Zhang H., Ferrara K.W., Wachsmann-Hogiu S., Li T., Airhart S., de Vere White R., Lam K.S, & Pan C.X. (2016). Novel Theranostic Nanoporphyrins for Photodynamic Diagnosis and Trimodal Therapy for Bladder Cancer. Biomaterials, 104, 339-351.

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